The present invention relates to vaccine adjuvants.
Vaccination has been chiefly responsible for the eradication of smallpox in man, and the control of numerous other infective agents in both animals and man. Traditionally vaccines have been whole organisms which are either attenuated, or killed. With the advent of recombinant DNA technology and increased understanding of immunology there has been much progress in producing sub-unit vaccines. Such vaccines are potentially free of problems associated with the traditional vaccines.
However, these new generation of vaccines are, on the whole, weakly immunogenic and thus require the presence of adjuvants (i.e. an agent that augments specific immune responses).
Materials having adjuvant activity are well known. Currently, however, Alum (Al(OH)3), and similar aluminium gels are the only adjuvants licensed for human use. The adjuvant activity of alum was first discovered in 1926 by Glenny (Chemistry and Industry, Jun. 15, 1926; J. Path. Bacteriol, 34, 267). Other materials are also known to have adjuvant activity, and these include: Freund""s complete adjuvant, a water-in-mineral-oil emulsion which contains killed, dried mycobacteria in the oil phase; Freund""s incomplete adjuvant, a weaker formulation without the mycobacteria; saponin, a membrane active glucoside extracted from the tree Quillia saponaria; nonionic block copolymer surfactants, non metabolised synthetic molecules which tend to bind proteins to cell surfaces; ISCOMS, lipid micelles incorporating Quil A (saponin) which mimic, in physical terms, infectious particles; and muramyl dipeptide, a leukocyte stimulatory molecule that is one of the active components of killed mycobacteria.
With all of these agents toxicity, and unacceptable chronic reactions, depending on dose, are a feature which currently limit their use as potential alternatives to alum. Alum, on the other hand, will not stimulate cell-mediated immunity, and although having a broad spectrum of activity, is not effective in all potential vaccines, since in peptide vaccines, adsorption onto the alum may be poor due to the small size of the peptide. Occasionally, alum may induce the degradation of antigens by proteases with great efficiency. Thus it is apparent that there is a need for new adjuvants.
NAGO, a combination of neuraminidase and galactose oxidase, is known in the art to induce T lymphocyte proliferation by the induction of aldehydes on cell membranes, (J. Immunol, vol 115(4), p932-8).
The present inventors have now discovered that a combination of neuraminidase and galactose oxidase (NAGO) possesses potent adjuvant properties. NAGO has been found to be a non-reactogenic adjuvant of unprecedented potency in the induction of T-cell responses. In particular it was as effective or better than Freund complete adjuvant in the induction of cytotoxic T-cells induced with peptide but recognizing cells infected with live virus. It was also more effective than Freund complete adjuvant in priming T-cells to the envelope glycoprotein gp120 of human immunodeficiency virus. Strong adjuvant effects were exemplified with peptide and protein and polysaccharide antigens of bacterial, viral and protozoal origin. Local reactions produced by NAGO were very mild and were no different to, or less than, those induced by alhydrogel, the only adjuvant licensed for human use.
Accordingly the present invention provides a vaccine formulation comprising an antigenic component and, as an adjuvant component, neuraminidase and galactose oxidase. The invention also provides the use, as a vaccine adjuvant, of neuraminidase and galactose oxidase. A vaccine may therefore be prepared by formulating the antigenic component with, as adjuvant, neuraminidase and galactose oxidase.
It will of course be appreciated that galactose oxidase and neuraminidase may be obtained from any suitable source but preferably, galactose oxidase is isolated from Dactilylium dendroides. The galactose oxidase from Dactilylium dendroides (EC 1.1.3.9) may have an activity of between 200-900 units (or u) per mg protein. Neuraminidase is preferably isolated from Vibrio cholerae or Clostridium perfringens. The Vibrio material (EC 3.2.1.18) preferably has an activity of 25u per xcexcg of protein (defined by the commercial source, BDH, Poole, Dorset, GB). When isolated from Clostridium, the enzyme preferably has an activity of 150-400 units per mg protein, although a partially purified material of 1 unit per mg solid has also shown to be effective. Both enzymes are commercially available from, for example, Sigma Chemical Company, Poole, Dorset, GB.
Preferably the ratio of neuraminidase to galactose oxidase in terms of units of activity is from 1:2 to 1:10 but optimally is about 1:5. The amount of NA per 100 xcexcl of material for injection may be from 0.05 to 12u, preferably from 0.2 to 1.2u. The amount of GO per 100 xcexcl of material for injection may be from 0.1 to 25u, preferably from 2 to 8u. The optimal amount is 1 u NA and 5u GO per 100 xcexcl of material for injection.
Antigens which may be particularly useful in a vaccine formulation include peptide, protein and carbohydrate antigens. The antigens may be bacterial, fungal, protozoal or viral antigens. The antigens may be subunit antigens from influenza (J. Immunol. 143 p3007, 1989), subunit antigens from human immunodeficiency virus (Lancet 335 p1081, 1990), such as gp 120, and subunit antigens from hepatitis virus (Lancet 335 p1142, 1990). Heat killed or otherwise attenuated whole organism vaccines (Lancet 335 p898, 1990) would also be suitable for use with NAGO. Other examples include antigens from polio virus, cytomegalovirus, herpes simplex viru""s, respiratory syncytial virus, rhinovirus, and Epstein Barr virus. Examples of animal viruses that would be compatible with NAGO include rabies, foot and mouth disease, equine ""flu, feline immunodeficiency and feline leukaemia virus (Lancet 335 p587, 1990).
Bacterial antigens such as from the following may advantageously be included in a vaccine formulation according to the present invention: B. pertussis, C. tetani, E. coli, C. diphtheriae, P. Aeruginosa, V. cholerae, H. influenzae, N. meningitidis, S. pneumoniae, N. gonorrhea and others with a suitable protein component or polysaccharide associated with a protein (Bacterial Vaccines, R. Germanier, ed., Academic Press Inc. New York, 1984).
In particular in relation to B. pertussis, the following components have been recognised as antigenic elements which may be used individually or in combination, when adjuvanted with NAGO, to provide an effective acellular vaccine against B. pertussis infections: filamentous haemagglutinin (FHA), P.69 (pertactin) and pertussis toxin (LPF) (Bacterial Vaccines, R. Germainier, ed. Chaper 3, Pertussis. Academic Press, New York, 1984). Antigens from parasites such as P. falciparum and L. major (Lancet 335 p1263, 1990) may also be adjuvanted by NAGO. Such an antigen is the principal malarial merozoite surface antigen.
Whilst alum will produce an adjuvant effect, our experiments show that NAGO is substantially more effective in producing antibody response in vivo. The potency of NAGO as an adjuvant raises the possibility of provoking undesirable, and unacceptable auto-immune responses to self-antigens. However, our experiments show this is highly unlikely since NAGO does not contravene the rules of genetically determined non-responsiveness. Thus, with influenza peptides of known MHC restrictions in mice, responses with NAGO as adjuvant occur when the MHC haplotype is permissive but not when it is non-permissive for a given peptide, even though regional lymph node lymphocyte numbers are increased in response to NAGO in the nonpermissive case.
The vaccine formulation may also comprise a suitable carrier, typically a conventional carrier for a vaccine for injection. NAGO is principally used in an aqueous diluent which may contain soluble or particulate antigens either alone or associated with a lipid carrier. NAGO may also be incorporated into water-in-oil emulsions and/or vehicles containing additional immunostimulatory elements such as muramyl dipeptide. Suitable amounts of vaccine formulations for injection into a patient are from 200 xcexcl to 2ml and typically 500 xcexcl (subcutaneous or intradermal for the smaller volumes, intramuscular for the larger).
The following examples serve to illustrate the present invention, but are not intended as a limitation thereof.